The study of rare genetic disorders of cancer susceptibility, such as Fanconi anemia (FA) have led to seminal advances in cancer biology as a whole. The hallmark of FA is genomic instability and DNA damage hypersensitivity, but the normal function of the FA pathway or mechanism leading to cancer in the affected patients remains unknown. FA is accounted for by multiple complementation groups corresponding to at least 8 cloned genes whose encoded proteins contain few functional motifs. What is known is that the FA pathway interacts with other more defined pathways involved in cellular response to DNA damage, such as ATM and BRCA1. The Kupfer laboratory has established that FA proteins such as FANCA and FANCG phosphorylated, that the FA core complex (composed of at least 5 FA proteins) localizes to at least 3 subcellular compartments, including chromatin, and that the core complex binds to additional non-characterized proteins. This proposal centers on the analysis of posttranslational modifications of the FA proteins FANCA and FANCG and on detecting new binding proteins and determining their function. This would shed light on the long-term goal of the Kupfer laboratory: define the mechanisms of genomic instability caused by FA as well as understand the normal function of the FA proteins. In this proposal the experiments proposed address the central hypothesis: that the phosphorylation of FANCA and FANCG regulate proper localization to chromatin and that a novel FA binding protein, RBM10, has RNA binding activity critical for this localization. The work proposed will 1) determine the cause and effect of FANCA and FANCG phosphorylation, 2) analyze RBM10 and RNA function in the FA core complex, and 3) focus on purifying additional FA core complex and FANCD2 binding proteins, especially from chromatin. This research is significant because it sheds light on a mechanism of genomic instability at the level of the functional genetic unit of the cell: chromatin. Because the response to DNA damage involves numerous intersecting pathways, defining the function of FA proteins will allow us to link these disparate pathways in a mechanism explaining cancer formation.